Gene expression is controlled at many different steps in the pathway from DNA to RNA to protein. Because aberrant gene expression can lead to a disease state, such as cancer, genes must be tightly regulated to ensure they are expressed at the correct time, place and level. While most efforts have been aimed at understanding transcriptional regulation of gene expression (i.e., DNA to RNA) and its contribution to disease, regulation at other levels such as mRNA translation (i.e., RNA to protein) or RNA stability remains less well understood. It is only recently that research into post-transcriptional mechanisms of gene expression has uncovered that regulation of mRNA translation, or translational control, is a critical checkpoint in gene expression linked to a variety of disease processes (Cazzola and Skoda, Blood 95: 3280-3288, 2000).
Translational control occurs in virtually all cell types and species where it contributes to such diverse processes as cell-cycle control, learning and plasticity in neurons, and red blood cell differentiation, among many others. Because translational control enables a cell to increase the concentration of a protein very rapidly, this mechanism of control is especially suited for the regulation of genes that are involved in cell proliferation and damage control. Regulation of gene expression at the level of mRNA translation is also particularly important in cellular responses to development or environmental stimuli—such as nutrient levels, cytokines, hormones, and temperature shifts, as well as environmental stresses—such as hypoxia, hypocalcemia, viral infection, and tissue injury. Translational control can be either global, affecting all the mRNAs in a cell, or specific to a single or subset of mRNAs.
The typical mRNA contains a 5′ cap, a 5′ untranslated region upstream of a start codon (5′ UTR), an open reading frame, also referred to as coding sequence, that encodes a functional protein, a 3′ untranslated region (3′ UTR) downstream of the termination codon, and a poly(A) tail. The key mediators of translational control are typically found in the 5′ and 3′ untranslated regions of mRNA transcripts, although the possibility of regulatory sequences mapping even to the coding sequence itself cannot be excluded. Much like the linear array of amino acids in proteins, these single-stranded regions of RNA can fold into complex three-dimensional structures consisting of local motifs such as hairpins, stem-loops, bulges, pseudoknots, guanosine quartets, and turns (for reviews see Moore, Ann. Rev. Biochem. 68:287-300, 1999; Gallego and Varani, Acc. Chem. Res. 34:836-843, 2001). Through interactions with regulatory proteins, such structures can be critical to the activity of the nucleic acid and dramatically affect the regulation of mRNA translation.
Because the sequences of an mRNA often contain critical regulatory elements which influence translational efficiency, compounds that are able to modulate the effect of the regulatory RNA sequence would be highly useful in therapeutic applications that seek to up- or downregulate the expression of a gene. Current approaches for blocking the function of target nucleic acids include the use of duplex-forming antisense oligonucleotides (Bennett and Cowsert, Biochem. Biophys. Acta 1489 (1): 19-30, 1999), peptide nucleic acids (“PNA”; Gambari, Curr. Pharm. Des. 7 (17): 1839-1862, 2001; Nielsen, Curr. Opin. Struct. Biol. 9 (3): 353-357, 1999; Nielsen, Curr. Opin. Biotechol. 10 (1): 71-75, 1999) and locked nucleic acid (“LNA”; Braasch & Corey, Chem. Biol. 8 (1): 1-7, 2001; Arzumanov et al., Biochemistry 40 (48): 14645-14654, 2001), which bind to nucleic acids via Watson-Crick base-pairing. However, the dependence on the native three-dimensional structural motifs of single-stranded stretches for regulatory functions can preclude the use of general, simple-to-use, sequence-specific recognition rules to design complementary agents that bind to these motifs.
Previous efforts to identify compounds or agents that recognize regulatory RNA elements have primarily focused on characterizing regulatory proteins that bind to a particular regulatory mRNA sequence, and on elucidating molecular mechanisms by which the protein-mRNA complex exerts its effect on translational control before identifying potential modulators. A major disadvantage of such approaches is the lengthy and laborious procedure required to isolate and identify proteins that bind to specific mRNA regulatory sequences. In addition to isolating the proteins that bind to regulatory mRNA sequences, these approaches have also either required the labeling of particular proteins or RNAs or depended on the linkage of the RNA regulatory sequence to a reporter, or a combination thereof. All these are time-consuming and laborious procedures that require a series of complex laboratory manipulations and often deliver false positive results. There is thus a need for a simplified method to identify modulators of translational control of gene expression that eliminates the requirement for a series of intermediate steps and yields a direct functional readout.